The role of bovine adenovirus (BAdV)-3 protein pVIII in virus replication
Bovine adenovirus (BAdV)-3 is a non-enveloped icosahedral DNA virus, which replicates in the nucleus of infected cells, and is being developed as a vector for vaccination for humans and animals. The genome of BAdV-3 is organized into early, intermediate and late genes and it has thirty three predicted open reading frames (Reddy et al., 1998). The late region of BAdV-3 is divided into seven families (L1-L7) (Reddy et al., 1998). One of the proteins expressed in the L-6 region encodes a protein called pVIII, which is a minor capsid protein connecting the core with the inner surface of the capsid. The objective of the current study was to characterize pVIII protein of BAdV-3 and to examine its role in the life cycle of BAdV-3. Anti-pVIII serum detected a protein of 24 kDa at 12-48 hr post infection and an additional protein of 8 kDa at 24-48 hr post infection. While a 24 kDa protein is detected in empty capsids, only the C-terminal cleaved protein of 8 kDa is detected in the mature virion suggesting that amino acids 147-216 of conserved C- terminus of BAdV-3 pVIII are incorporated in mature virions. The pVIII protein predominantly localizes to the nucleus of BAdV-3 infected cells utilizing the classical importin α /β dependent nuclear import pathway. Analysis of mutant pVIII demonstrated that amino acids 57-72 of the conserved N-terminus bind to importin α-3 with high affinity and are required for the nuclear localization. Detection of hexon associated with both, precursor (24 kDa) and cleaved (8 kDa) form of pVIII suggests that the C-terminus of pVIII interacts with Hexon. Based on yeast II hybrid screening assay, we identified the cellular protein DDX3 as an interacting protein partner of pVIII. Earlier, targeting of DDX3 by few viral proteins has defined its role in mRNA transport (Yedavalli et al., 2004) and induction of interferon production (Schroder et al., 2008; Wang et al., 2009). Here, we provide evidence regarding the involvement of DDX3 in cap dependent cellular mRNA translation and show that targeting of DDX3 by the adenovirus pVIII protein abolishes cap-dependent mRNA translation function of DDX3 in virus infected cells. Adenovirus late protein pVIII interacts with DDX3 in transfected and bovine adenovirus (BAdV-3) infected cells. pVIII inhibited capped mRNA translation in-vitro and in-vivo by limiting the amount of DDX3 and eIF3. Diminished amount of DDX3 and eIFs including eIF3, eIF4E and PABP were present in cap binding complex in BAdV-3 infected or pVIII transfected cells with no trace of pVIII in the cap binding complex. The total amount of eIFs appeared similar in uninfected or BAdV-3 infected cells. The co-immunoprecipitation experiments indicated the absence of direct interaction between pVIII and eIF3, eIF4E or PABP. These data indicate that interaction of pVIII with DDX3 depletes eIF3, eIF4E and PABP from the cap-binding complex. We conclude that DDX3 promotes cap-dependent cellular mRNA translation and BAdV-3 pVIII inhibits translation of capped cellular mRNA by excluding functional cap-binding complex from the capped cellular mRNA. BAdV-3 infection of DDX3 positive cells significantly inhibits cellular protein synthesis at late times post-infection. Interestingly, knockdown of DDX3 resulted in significant reduction in virus yield and expression of BAdV-3 late proteins at late times post-infection. Our results suggest that selective translation of BAdV-3 late mRNAs observed at late time post-infection of DDX3 positive cells is abrogated in DDX3 knock down cells. Moreover, the reduction in the extent of protein synthesis is evidenced by less functional 80S and polysomes in pVIII expressing plasmid transfected cells. Alternatively, DDX3 and pVIII binds to BAdV-3 tripartite leader (TPL) and the translation of mRNAs containing TPL at their 5’ ends is enhanced in the presence of pVIII and DDX3 proteins. From this observation, we concluded that pVIII and DDX-3 might promote the translation of late viral mRNAs by interacting with TPL.
BAdV-3, pVIII, Nuclear import, DDX3, mRNA translation, TPL
Doctor of Philosophy (Ph.D.)